The sensitivity of cultured human cells to the lethal effects of ionizing radiation (IR) is a complex trait that is contributed to by multiple genes and their products functioning within various pathways involved in DNA damage sensing and response. The focus of our studies supported by this grant has been to identify genes in human populations that contribute to radiation sensitivity with the long-term goals of (1) understanding the relationship between exposure to an environmental mutagen (IR) and cancer development, and (2) developing markers for radiation sensitivity that would allow us to better tailor radiation therapy protocols to individual patient sensitivities. This approach has led us to study patients that represent the extremes of radiation sensitivity in human populations, patients with the inherited disorders Ataxia- telangiectasia (A-T) and Nijmegen breakage syndrome (NBS) where the radiation sensitive phenotype is inherited as a recessive trait, allowing the mapping and positional cloning of the responsible genes. In the previous funding period, we successfully identified the gene for NBS and its protein product, nibrin, and elucidated the biochemical connection between A-T and NBS by demonstrating that nibrin is a substrate for the ATM kinase. In this application we propose to extend our studies of A-T and NBS with 2 general approaches. In the first, we will focus on the function of nibrin. We will map functional domains on the protein by site-specific mutagenesis, identify other proteins with which it interacts by following leads from a completed yeast two-hybrid screen, and develop a mouse model to explore the function of nibrin in tissues and at developmental stages not accessible in human patients. In the second approach, we will use transcript profiling with microarrays in cell lines with specific defects in DNA damage response pathways to identify new molecules in these pathways and to develop a "fingerprint" of transcript expression associated with radiation sensitivity. Finally, we will search for additional genes involved in radiation sensitivity in a collection of NBS families we have identified that lack mutations in the NBS1 gene, indicating that NBS is a heterogeneous disorder. These proposed studies should allow us to extend the insights gained from the identification of genes responsible for rare disorders characterized by radiation sensitivity into a more detailed understanding of the biochemical pathways activated in human cells in response to ionizing radiation.